Development of an Rev-independent, minimal simian immunodeficiency virus-derived vector system.

Lentiviral vectors are attractive candidates for gene therapy because of their ability to integrate into nondividing cells. To date, conventional HIV-1-based vectors can be produced at higher titers, but concerns regarding their safety for human use exist because of the possibility of recombination leading to production of infectious virions with pathogenic potential. Development of lentivirus vectors based on nonhuman lentiviruses constitutes an active area of research. We described a novel HIV-SIV hybrid vector system in which an HIV-1-derived transfer vector is encapsidated by SIVmac1A11 core particles and pseudotyped with VSV glycoprotein G. In an effort to further develop this vector system, we modified the packaging plasmid by deletion of the SIV accessory genes. Specifically, versions of the packaging plasmid (SIVpack) lacking vif, vpr, vpx, and/or nef were constructed. Our results indicate that, as with HIV-1-based packaging plasmids, deletion of accessory genes has no significant effect on transduction in either dividing or nondividing cells. The SIV packaging plasmid was also modified with regard to the requirement for RRE and rev. Deletion of the RRE and rev from SIVpack led to dramatic loss of transduction ability. Introduction of the 5' LTR from the spleen necrosis virus to packaging plasmids lacking RRE/Rev was then sufficient to fully restore vector titer. A minimal SIV transfer vector was also developed, which does not require RRE/Rev and exhibits no reduction in transduction efficiency in two packaging systems. The SIV-based vector system described here recapitulates the biological properties of minimal HIV-1-derived systems and is expected to provide an added level of safety for human gene transfer. We suggest that the SIV-derived vector system will also be useful to deliver anti-HIV-1 gene therapy reagents that would inhibit an HIV-1-derived vector.

Preparation and induction of immune responses by a DNA AIDS vaccine.

In an effort to evaluate the feasibility of developing a safe DNA vaccine for acquired immunodeficiency syndrome (AIDS), we have prepared a plasmid-based immunogen modeled after a naturally occurring noninfectious mutant of the simian immunodeficiency virus (SIV). The mutant SIV genome produces defective virus particles that are noninfectious in vitro and nonpathogenic in vivo in rhesus macaques. Analysis of the mutant genome revealed a 1.6 kb deletion that is in frame and spans integrase, vif, vpx, and most of vpr and results in a pol/vpr gene fusion. This deletion was introduced into the parental pathogenic molecular clone and the U3 region of the 5' LTR was replaced with a cytomegalovirus promoter to produce a candidate DNA vaccine, pIV. After transfection with this plasmid, SIV gag and envelope proteins are expressed and properly processed in vitro. When injected into rabbits, pIV elicited an antibody response to SIV gp130 envelope glycoprotein with titers reaching 1:2048, and a strong lymphoproliferative response to SIV gp130 and whole SIV. The potential to produce defective virus particles in vivo without integrating into the host genome should result in both a strong humoral and cellular immune response in rhesus macaques. In addition, this approach offers a safe alternative to live attenuated vaccines and DNA vaccines that are capable of integration.

The kinetics of specific immune responses in rhesus monkeys inoculated with live recombinant BCG expressing SIV Gag, Pol, Env, and Nef proteins.

Development of an effective preventive or therapeutic vaccine against HIV-1 is an important goal in the fight against AIDS. Effective virus clearance and inhibition of spread to target organs depends principally on the cellular immune response. Therefore, a vaccine against HIV-1 should elicit virus-specific cytotoxic lymphocyte (CTL) responses to eliminate the virus during the cell-associated stages of its life cycle. The vaccine should also be capable of inducing immunity at the mucosal surfaces, the primary route of transmission. Recombinant Bacille Calmette-Guérin (BCG) expressing viral proteins offers an excellent candidate vaccine in view of its safety and ability to persist intracellularly, resulting in the induction of long-lasting immunity and stimulation of the cellular immune response. BCG can be administered orally to induce HIV-specific immunity at the mucosal surfaces. The immunogenicity of four recombinant BCG constructs expressing simian immunodeficiency virus (SIV) Gag, Pol, Env, and Nef proteins was tested in rhesus macaques. A single simultaneous inoculation of all four recombinants elicited SIV-specific IgA and IgG antibody, and cellular immune responses, including CTL and helper T cell proliferation. Our results demonstrate that BCG recombinant vectors can induce concomitant humoral and cellular immune responses to the major proteins of SIV.

Cell tropism of the simian foamy virus type 1 (SFV-1).

Several cell lines representing different species and cell types were tested for simian foamy virus type 1 (SFV-1) infection. SFV-1 infections were monitored by polymerase chain reaction, reverse transcriptase, cytopathology, and immunofluorescent assays. All cells tested were permissive for SFV-1, demonstrating that SFV-1 has a broad host range with respect to species and cell types. Infected fibroblasts, epithelial cells, and neural cells all showed extensive cytopathology that is characteristic of foamy virus infection. No cytopathology was induced in T cell-derived Jurkats and Hut-78 cell lines. The cytopathic effects in B and macrophage originated cells were delayed by several days. Cytopathology in these cell lines was not as dramatic as seen in the infected fibroblast and epithelial cells. The reverse transcriptase values in the SFV-1 infected lymphoid and macrophage cell lines were severalfold lower than that of the fibroblasts and epithelial cells. Therefore, SFV-1 appears to establish a low level persistent infection in lymphoid and macrophage cell lines.

Genetic and biological comparisons of pathogenic and nonpathogenic molecular clones of simian immunodeficiency virus (SIVmac).

Simian immunodeficiency virus (SIV) is a designation for a group of related but unique lentiviruses identified in several primate species. A viral isolate from a rhesus macaque (i.e., SIVmac) causes a fatal AIDS-like disease in experimentally infected macaques, and several infectious molecular clones of this virus have been characterized. This report presents the complete nucleotide sequence of molecularly cloned SIVmac1A11, and comparisons are made with the sequence of molecularly cloned SIVmac239. SIVmac1A11 has delayed replication kinetics in lymphoid cells but replicates as well as uncloned SIVmac in macrophage cultures. Macaques infected with virus from the SIVmac1A11 clone develop antiviral antibodies, but virus does not persist in peripheral blood mononuclear cells and no disease signs are observed. SIVmac239 infects lymphoid cells, shows restricted replication in cultured macrophages, and establishes a persistent infection in animals that leads to a fatal AIDS-like disease. Both viruses are about 98% homologous at the nucleotide sequence level. In SIVmac1A11, the vpr gene as well as the transmembrane domain of env are prematurely truncated, whereas the nef gene of SIVmac239 is prematurely truncated. Sequence differences are also noted in variable region 1 (V1) in the surface domain of the env gene. The potential implications of these and other sequence differences are discussed with respect to the phenotypes of both viruses. This animal model is critically important for investigating the roles of specific viral genes in viral/host interactions that cannot be studied in individuals infected with the human immunodeficiency virus (HIV).